A Preliminary Study on Anti-inflammatory and Analgesic Effects of Millettia speciosa and Tinpspora sinensis and Their Compatibility

Shibo ZHAO, Suoyi HUANG2, 3*, Xinpeng CHEN, Huanhui DENG, Zhi PAN, Kairen XIE, Haoming YU, Fen MO

1. College of Clinical Medicine, Youjiang Medical University for Nationalities, Baise 533000, China; 2. College of Pharmacy, Youjiang Medical University for Nationalities, Baise 533000, China; 3. Youjiang Medical University for Nationalities, Key Laboratory of Guangxi Universities on National Medicine in Youjiang River Basin, Baise 533000, China

Abstract [Objectives] To study the anti-inflammatory and analgesic effects of Millettia speciosa and Tinpspora sinensis and their compatibility. [Methods] (i) In the glacial acetic acid writhing experiment, 60 SPF-grade Kunming mice were adopted, and the mice were randomly divided into 6 groups at male-female ratio of 1∶1, namely, the blank control group, M. speciosa group, T. sinensis group, M. speciosa compatible with T. sinensis group at the ratio of 1∶2 (expressed as 1∶2 compatibility group), M. speciosa compatible with T. sinensis group at the ratio of 1∶1 (expressed as 1∶1 compatibility group), and M. speciosa compatible with T. sinensis group at the ratio of 2∶1 (expressed as 2∶1 compatibility group), 12 mice for each group. Mice of the experimental groups were administered at a dose of 20 mL/kg, and the corresponding concentration of the Chinese medicine extract was given at 1 g/mL. The control group was administered with an equal volume of 0.9% physiological saline, and was intragastrically administered once every 24 h for 14 d. After intragastric administration for one hour on day 14, intraperitoneal injection of 0.5% glacial acetic acid solution was performed to induce pain. (ii) In the hot plate experiment, 60 Kunming female SPF mice were adopted, grouped, intragastrically administered with the same glacial acetic acid writhing experiment for 14 d. After intragastric administration for one hour on day 14, the mice were placed on a hot plate apparatus at (55±0.5) ℃. to measure the time of licking their hind feet. (iii) In the anti-inflammatory experiment, 60 Kunming SPF mice were adopted, grouped, intragastrically administered with the same glacial acetic acid writhing experiment for 14 d. After intragastric administration for one hour on day 14, xylylene was administered to the left ears of mice at a dose of 50 μL/piece to induce inflammation. [Results] (i) In the glacial acetic acid writhing experiment, compared with the blank control group, the experimental group showed analgesic effects. Specifically, M. speciosa group, T. sinensis group, 1∶2 compatibility group, 1∶1 compatibility group, 2∶1 compatibility group showed significant effect (P<0.05), the writhing inhibition rate was 17.65%, 20.59%, 29.41%, 26.47%, and 44.12%, respectively, and 2∶1 compatibility group showed the most significant analgesic effects. (ii) In the hot plate experiment, compared with the control group, all experimental groups showed analgesic effect. Specifically, M. speciosa group, T. sinensis group, 1∶2 compatibility group, 1∶1 compatibility group, 2∶1 compatibility group showed significant effect (P<0.05), the pain threshold improvement rates were 16.13%, 14.55%, 14.96%, 29.95%, and 58.68%, respectively, and 2∶1 compatibility group showed the most significant analgesic effect. (iii) In the anti-inflammatory experiment, the swelling degree of the 1∶2 compatibility group was significantly different from that of the blank control group, M. speciosa group, T. sinensis group (P<0.05). and 1∶2 compatibility group showed the most significant anti-inflammatory effect. [Conclusions] M. speciosa, T. sinensis, and their compatibility had anti-inflammatory and analgesic effects. The 2∶1 compatibility group had the best analgesic effects, and 1∶2 compatibility group had the best anti-inflammatory effects.

Key words Millettia speciosa, Tinpspora sinensis, Compatibility, Anti-inflammatory effects, Analgesic effects

1 Introduction

MillettiaspeciosaChamp. is a plant of theMillettiagenus belonging to the Leguminosae family, and its dried tubers are widely applied as medicines[1].M.speciosa, sweet in taste and neutral in nature, has functions of tonifying deficiency and moistening lung, strengthening sinews and activating collaterals. Clinically, it has a certain effect on chronic diseases such as lumbar muscle strain, rheumatoid arthritis, tuberculosis, chronic bronchitis,etc.[2].Tinosporasinensis(Lour.) Merr. is a plant ofTinosporaMiersgenus belonging to the Menispermaceae family[3]. Its dried tubers are widely applied as medicines. It is bitter in taste and cool in nature, and has the functions of relaxing sinews and activating collaterals, and dispelling wind and relieving pain[4]. Clinically, it is mainly applied for trauma diseases such as fractures, joint stiffness, rheumatism and pain, lumbar muscle strain, and bruises[5]. Some researchers have found that bothM.speciosaandT.sinensishave anti-inflammatory and analgesic effects[6-7]. In studying the effect ofM.speciosaon urate-induced synovial cell inflammation in mice, Huang Huietal.[8]confirmed thatM.speciosacould relieve urate-induced synovial cell inflammation in mouse models. Zeng Congyanetal.[9]have confirmed thatT.sinensishas a certain therapeutic effect on rats with adjuvant arthritis.

The chemical components and pharmacology ofM.speciosaandT.sinensishave been extensively studied. Many scholars have studied them in various fields. The pharmacological research ofM.speciosaandT.sinensishas been deepened both at home and abroad, but there is no relevant report on the compatibility ofM.speciosaandT.sinensis. The anti-inflammatory and analgesic effects ofM.speciosaandT.sinensison mice and whether the two have a good synergistic effect. This experiment is intended to provide a theoretical basis for further expanding the application and development ofM.speciosaandT.sinensisand their clinical application.

2 Materials and methods

2.1InstrumentsTWCL-T 5000 magnetic stirrer (Shanghai Biaohe Instrument Co., Ltd.); JMF-320G multi-stage flash evaporator (Henan Zhijing Biological Technology Co., Ltd.); DLSB-5 low-temperature cooling liquid circulating pump (Zhengzhou Great Wall Scientific Industrial & Trade Co., Ltd.); RB-200 intelligent hot plate apparatus (Chengdu TechMan Sofware Co., Ltd.); JSC electronic balance scale CN-BH (Fuzhou Kedi Electronic Technology Co., Ltd.); FA1204B electronic balance (Shanghai Techcomp Instrument Co., Ltd.).

2.2ExperimentalmaterialsFeed for clean-grade laboratory mice feed, produced by Jiangsu Xietong Medical Bioengineering Co., Ltd.;M.speciosa, produced by Guangxi Yinuo Shangpin TCM Decoction Pieces Co., Ltd., batch No.170801;T.sinensis, produced by Guangxi Yinuo Shangpin TCM Decoction Pieces Co., Ltd., batch No.171101.

2.3ExperimentalanimalsandgroupsA total of 180 SPF Kunming mice, 120 female and 60 male, weighed 18-22 g, and their week age was unknown. They were provided by Laboratory Animal Center of Youjiang Medical University for Nationalities, license No.SCXK Gui 2012-0003.

The male and female mice of each group were randomly assigned 1∶ 1, with 12 mice in each group and cages were divided by sex, with 6 mice in each cage. They were marked with 5% picric acid as 6 groups using the random number table method: the blank control group,M.speciosagroup,T.sinensisgroup,M.speciosacompatible withT.sinensisgroup at the ratio of 1∶2 (expressed as 1∶2 compatibility group),M.speciosacompatible withT.sinensisgroup at the ratio of 1∶1 (expressed as 1∶1 compatibility group), andM.speciosacompatible withT.sinensisgroup at the ratio of 2∶1 (expressed as 2∶1 compatibility group).

2.4IdentificationofmedicinalmaterialsThe medicinal materials were identified by Professor Huang Suoyi from Scientific Experiment Center of Youjiang Medical University for Nationalities as dried tubers ofMillettiaspeciosa(Millettiagenus, Leguminosae family) and dried stems ofTinpsporasinensis(Lour.) Merr (TinosporaMiersgenus, Menispermaceae family).

2.5MedicineextractionTook 500 g ofM.speciosapowder, soaked in 8 times the volume of distilled water for 1 h, heated to boil for 2 h, filtered out the solution while it was still hot and added 6 times the volume of distilled water to boil for 1 h, then combined the filtrate and concentrated to a concentration of 1 g/mL. Took 500 g ofT.sinensispowder, added 10 times the volume of distilled water to soak for 2 h and heat for 1 h, filtered out the solution while it was still hot and added 8 times the distilled water. In the third time, added 5 times the distilled water, heated for 1 h, and combined the filtrate and concentrated to the same concentration asM.speciosa. The 1∶1 compatibility group was prepared by mixing the preparedM.speciosaextract andT.sinensisextract in a volume ratio of 1∶ 1. The 1∶2 compatibility group and the 2∶1 compatibility group were prepared by the same method as the 1∶1 compatibility group. After all the medicines were prepared, put them in a refrigerator at 4 ℃ for experiment.

2.6AnimalgroupsanddrugadministrationThe body mass of mice were measured after one week of adaptive feeding. In order to ensure the accuracy of dose of intragastrical administration, the body mass was measured every 3 d during the experiment. In the experiment, each group was given the Chinese medicine extract according to the body mass of 20 mL/kg, that is, a dose of 20 g/kg (the mass concentration of the medicine solution was 1 g/mL, 1 mL of the medicine solution contained 1 g of Chinese medicine, and the intragastric administration volume was 20 mL/kg, the dose was 20 g/kg), the blank control group was the normal saline group, given 0.9% physiological saline at the equal volume. Other groups were given corresponding Chinese medicine extracts. One time daily, continuously intragastric administration for 14 d. Each animal experiment complied with the ethical requirements of animal experiment.

2.7AnalgesicexperimentofM.speciosaandT.sinensisandtheircompatibilityonwrithingreactioninducedbyglacialaceticacidinmiceSixty SPF mice were selected. The ratio of male to female, grouping, administration and body mass measurement were the same as in Section2.6. Thirty minutes after the last administration, each mouse was injected intraperitoneally with 20 mL/kg of 0.5% glacial acetic acid solution, and the first writhing time and the number of writhing reactions of the mice were recorded within 30 min after the acetic acid injection, and the writhing inhibition rate was calculated (note: when the mice had abdominal invaginations, trunk and hindlimb extensions, and raised hips, it means that there is writhing reaction). The writhing inhibition rate was calculated using the following formula.

Writhing inhibition rate = (Average writhing times of the control group-Average writhing times of drug groups)/Average writhing times of the control group × 100%.

2.8AnalgesicexperimentofM.speciosaandT.sinensisandtheircompatibilityonhotplate-inducedpaininmiceSixty female mice were selected. Grouping, administration and body mass measurement were the same as in Section2.6. Before the beginning of the experiment and 1 h after the last administration, placed the mice on a hot plate[set temperature at (55±0.5) ℃] and then immediately started timing, made a record of the time required for the mice to contact the instrument to lick the hind feet as the pain threshold for this mouse. After the measurement, calculated the pain threshold improvement rate using the following calculation formula.

Pain threshold improvement rate = (Average pain threshold after administration-Average pain threshold before administration)/Average pain threshold before administration × 100%.

2.9EffectsofT.sinensisandM.speciosaonearswellinginmiceWith reference to the experiment of mouse ear swelling induced by xylene carried out by Yu Huangheetal.[10], 60 mice were selected, and the ratio of male to female, grouping, administration and body mass measurement were the same as in Section2.6. One hour after the last administration, xylene was applied to the left ear of the mouse at 50 μL/piece, and the right ear was used as a control. After 15 min, killed the mice through breaking the neck, and punches were punched along the same part of the left and right auricles with a 6 mm diameter punch, the mass of both left and right ears were weighed separately, and the difference between the mass of the left and right ears was the degree of ear swelling, and then calculated the swelling inhibition rate.

Swelling inhibition rate=(Average value of the model group-Average value of the dry administration group)/Average value of the model group×100%.

3 Results and analysis

3.1AnalgesiceffectsonwrithingreactioninducedbyglacialaceticacidinmiceT.sinensisandM.speciosaand their compatibility have synergistic effects on the first time of writhing, the number of writhing in 30 min, and the writhing inhibition rate in mice. Especially, the inhibition effect in the 2∶1 compatibility group was significant, and the writhing inhibition rate reached 44.12%, indicating that compatibility ofM.speciosaandT.sinensisat the ratio of 2∶1 had excellent analgesic effect (Table 1).

GroupDose∥g/kgFirst writhing time∥sTimes of writhing within 30 min∥timesWrithing inhibition rate∥%Blank control-241±57.31234.750±7.615-M. speciosa20288±46.670a28.500±7.399a17.65T. sinensis20273±61.551a27.500±7.399a20.591∶2 compatibility20268±58.433ab24.311±7.506ab29.411∶1 compatibility20291±44.786a25.303±6.731ab26.472∶1 compatibility20342±72.628abc19.729±6.757abc44.12

Note: compared with the blank control group,adenotesP<0.05; compared with theM.speciosagroup,bdenotesP<0.05; compared with theT.sinensisgroup,cdenotesP<0.05.

3.2Effectsonpainreactionofmiceinducedbyhotplate

BothM.speciosaandT.sinensisand their compatibility had a synergistic effect on the time of licking hind feet and the pain threshold improvement rate of pain caused by hot plate. Especially, 2∶1 compatibility group had more significant effect than the single drug. Through analysis, it was found that the time of licking hind feet in the 2∶1 compatibility group was significantly improved compared with the blank control group,M.speciosagroup andT.sinensisgroup (P<0.05), and the pain threshold improvement rate reached 58.681%, indicating that compatibility ofM.speciosaandT.sinensisat the in ratio of 2∶1 had better analgesic effect (Table 2).

GroupDose∥g/kgBefore drug administration∥s14 d after drug administration∥sPain threshold improvement rate∥%Blank control-24.195±5.89924.806±4.733-M. speciosa2027.808±2.44032.292±2.37116.13T. sinensis2026.904±2.77330.818±6.24014.551∶2 compatibility2023.449±2.03938.407±2.439a14.961∶1 compatibility2029.185±1.629a41.661±3.238a29.952∶1 compatibility2026.801±1.81042.538±4.010abc58.68

Note: compared with the blank control group,adenotesP<0.05; compared with theM.speciosagroup,bdenotesP<0.05; compared with theT.sinensisgroup, c denotesP<0.05.

3.3Anti-inflammatoryeffectsonxylene-inducedearswellinginmiceThe acute inflammatory response of xylene-induced ear swelling in mice may involve the release of inflammatory mediators, such as serotonin or bradykinin[11]. BothM.speciosaandT.sinensisand their compatibility had a synergistic effect on the degree of ear swelling and swelling inhibition rate caused by xylene-induced ear swelling in mice. Data research showed that ear swelling in the three compatibility groups was inhibited by varying degrees. The 1∶2 compatibility group had the most significant effect (P<0.05) and had excellent anti-inflammatory effect, as listed in Table 3.

GroupDose∥g/kgLeft ear∥mgRight ear∥mgDegree of swelling∥mgSwelling inhibition rate∥%Blank control-3.127±2.3771.900±1.2951.327±1.453-M. speciosa202.600±1.0601.850±1.1830.733±0.859a44.76T. sinensis202.121±1.310a1.700±1.1990.550±0.694a58.551∶2 compatibility202.053±1.133ab1.871±1.4830.273±0.696abc79.941∶1 compatibility202.200±1.767ab1.883±1.3140.348±0.692ab73.782∶1 compatibility202.379±1.757a2.086±1.4070.375±0.780ab71.74

Note: compared with the blank control group,adenotesP<0.05; compared with theM.speciosagroup,bdenotesP<0.05; compared with theT.sinensisgroup,cdenotesP<0.05.

4 Discussions

Inflammation, as the most basic pathological process of many diseases, is mainly characterized by local microvascular response and exudation of blood components, and the local clinical features are redness, swelling, pain, and dysfunction[12]. The development of inflammation is generally divided into three stages. At the early stage, capillary vasodilation and hyperpermeability are the main types, platelet adsorption and leukocyte migration are at the middle stage, and granulation tissue hyperplasia appear at the later stage[13]. According to experimental research, Zheng Yuansheng[14]found that bovine polysaccharide can significantly reduce the xylene-induced local inflammation of mouse auricles. From this, it can be known that anti-inflammatory active components contained inM.speciosapolysaccharide also has a significant inhibitory effect on the exudation rate of inflammatory factors caused by xylene-induced ear swelling. Through experiment, Cao Zhifang[15]proved thatM.speciosapolysaccharide can reduce the level of cyclooxygenase-2 (COX-2), and its anti-inflammatory mechanism may be related to inhibition of cyclooxygenase-2 (COX-2) synthesis. According to the research by Liu Dandanetal.[16],M.speciosawater extract has obvious inhibitory effects on pain caused by physical and chemical factors, has certain peripheral analgesic and central analgesic effects, and can inhibit clinical inflammatory reaction induced pain. Xue Qiangetal.[17]found thatT.sinensiswater extract has an inhibitory effect on tissue swelling in mice. However, there are no related reports on the study of anti-inflammatory and analgesic mechanisms ofT.sinensis, so the synergistic mechanism of anti-inflammatory and analgesic needs to be further studied.

Pain is one of the most common clinical symptoms. Pain can be used as a warning of the body’s injury to cause the body’s defensive protective response[18]. The purpose of pain treatment is to reduce trauma, improve patient comfort, and improve quality of life[19]. The principle of the analgesic effect research experiment is to apply the stimulus response to the experimental animal to cause pain, observe the effect of the drug on the experimental animal’s pain response, and evaluate the efficacy of the drug[20]. Wu Fengrongetal.[21]found thatT.sinensisdecoction may have peripheral and central analgesic effects, and inhibit both acute and chronic pain to a certain extent.

Chemical components of traditional Chinese medicine are very complicated, and the role of pharmacologically active components is comprehensive. Some components have both therapeutic effects and certain toxicity[22]. Antipyretic and analgesic anti-inflammatory drugs are closely related to human life and are one of the most prescribed drugs in the world[23]. In this experiment, we made a preliminary study on the anti-inflammatory and analgesic effects ofM.speciosaandT.sinensisand their compatibility through glacial acetic acid writhing test, hot plate pain test and xylene-induced ear swelling test. The results indicated thatM.speciosa,T.sinensis, and their compatibility had anti-inflammatory and analgesic effects. The 1∶2 compatibility group had the best anti-inflammatory effects, and the 2∶1 compatibility group had the best analgesic effects This experiment is only a preliminary exploration experiment, to provide a new idea for further development of the medicinal value and clinical application ofM.speciosaandT.sinensisg. Further research is needed on the other synergistic effects, anti-inflammatory and analgesic action mechanisms and other clinical applications ofM.speciosa,T.sinensisand their compatibility.